Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │1
Standardization and Certification of the ADVIA Centaur
Vitamin D Total Assay
Authors:
James Freeman
Paul Sibley, PhD
Neil Parker, MSc
Ryan Spears
Kimberly S. Wilson
H. Roma Levy, MS
Affiliations:
Siemens Healthineers
511 Benedict Avenue
Tarrytown, NY 10591-5005
USA
Phone: + 914-631-8000
siemens.com/healthineers
Correspondence should be
addressed to:
Neil Parker, MS:
511 Benedict Avenue, Tarrytown,
NY 10591, USA
Tel: +1 (914) 524 2477
E-mail:
neil.np.parker@siemens-
healthineers.com
Running Title: ADVIA Centaur
Vitamin D Total Assay
Standardization/Certification
Keywords: Vitamin D, ADVIA
Centaur, Vitamin D Standardization
Program, VDSP; Vitamin D
Standardization Certification
Program, VDSCP; NIST-Ghent-
RMP; ID-LC/MS/MS
Abstract
Background: In recent years, the Siemens ADVIA Centaur®
Vitamin D Total assay was standardized for traceability to the
National Institute of Standardization and Technology (NIST)-
Ghent reference method procedure (RMP) used in the
Vitamin D Standardization Program (VDSP). Here, we
compare results from the newer ADVIA Centaur Vitamin D
assay to results obtained using several methods traceable to
the VDSP NIST-Ghent RMP. We also present results of
successful participation in the Centers for Disease Control and
Prevention (CDC) vitamin D certification program (VDSCP).
Methods: The ADVIA Centaur Vitamin D Total assay results
were compared to values assigned at Ghent University
(n = 122); values assigned by methods metrologically
traceable to the VDSP NIST-Ghent RMP (n = 177); RMP-
assigned values by the College of American Pathologists
(CAP) and Vitamin D External Quality Assessment Scheme
(DEQAS) survey (n = 30); and to a third-party VDSCP-
certified assay (n = 149). The CDC certification scheme
(VDSCP), which requires annual certification, was applied
over four consecutive years (2013–2017).
Results: Correlation between the standardized ADVIA
Centaur Vitamin D Total assay and the VDSP NIST-Ghent
RMP results was 99%; bias was –0.09%. Bias in comparison
to CAP and DEQAS samples was < 25% for 96% of samples.
Bias for CDC VDSCP certification samples was 0.3%, mean
CV was 5.5%. Correlation to the Esoterix CDC VDSCP-
certified ID-LC/MS/MS method was 95% for vitamin D total
and 97% for samples containing only 25(OH)D3.
Conclusions: The ADVIA Centaur Vitamin D Total assay
traceable to the VDSP NIST-Ghent RMP demonstrated good
performance and is acceptable for clinical use based on
imprecision, and by comparison to CAP and DEQAS surveys.
The assay is aligned to the VDSP NIST-Ghent RMP, and was
among the first automated immunoassays certified by the
CDC VDSCP. As of February 2017, the assay has achieved
VDSCP certification for four consecutive years.
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │2
1. Introduction
Assessing vitamin D levels by
measuring total 25-hydroxy vitamin D
(25[OH]D), i.e. combined 25[OH]D2 and
25[OH]D3) provides important clinical
information.
[The Institute of Medicine has defined
the risk of Vitamin D deficiency as a
25(OH)D concentration less than 12 ng/L
(30 nmol/L), whereas the Endocrine Society
has defined Vitamin D deficiency as a
25(OH)D concentration less than 20 ng/mL
(50 nmol/L).1,2
The Endocrine society
defines insufficiency as 20 to 30 ng/mL
(50 to 75 nmol/L)].2
Because 25(OH)D is a small steroid
hormone with a very high affinity for its
carrier protein (vitamin D-binding protein
[VDBP]), evaluation is a challenging process.
Several studies and national surveys have
noted substantial variability between methods
due to differences in manufacturers’
proprietary methods for releasing 25(OH)D
from VDBP, variability in the molar ratio of
recovered 25(OH)D2 and 25(OH)D3, and the
amount of epimeric forms detected—
especially 3-epi-25(OH)D3.3-8
Lack of assay
standardization to a single reference method
can pose a substantial impediment to clinical
assessment, especially if evaluation over time
and location utilizes different methods. Assay
variability has also affected efforts to
establish reliable clinical guidelines and
laboratory proficiency testing.
To address assay variability problems,
the National Institutes of Health (NIH) Office
of Dietary Supplements (ODS) instituted the
25(OH)D Vitamin D Standardization
Program (VDSP). As a collaborative effort of
the National Institute of Standards and
Technology (NIST), the Centers for Disease
Control and Prevention (CDC), and Ghent
University (Ghent, Belgium), laboratories and
manufacturers are provided with reference
materials and a reference protocol to
standardize the vitamin D measurement,
thereby improving diagnostic accuracy of
vitamin D regardless of assay method.9-13
To complement this program, manufacturers
and laboratories may participate in a
certification process administered by the
CDC: the Vitamin D Standardization-
Certification Program (VDSCP).
Siemens Healthineers (Tarrytown, NY,
U.S.), an early adopter of the VDSP,
standardized its ADVIA Centaur® Vitamin D
Total assay against the 25(OH)D VDSP
NIST-Ghent RMP using metrologically
traceable assay standards.
The goal of this study was to evaluate
the standardized ADVIA Centaur Vitamin D
Total assay performance using several
methods that include the VDSP NIST-Ghent
RMP method and methods metrologically
traceable to the NIST-Ghent VDSP-RMP,
and according to the specifications of the
CDC Vitamin D Standardization Certification
Program (VDSCP).
2. Materials and Methods
2.1. The VDSP NIST-Ghent RMP
The VDSP NIST-Ghent RMP is an
isotope dilution, liquid chromatography,
tandem mass spectrometric (ID-LC/MS/MS)
method that measures only 25(OH)D2 and
25(OH)D3 and does not measure either 24, 25
dihydroxyvitamin D or 3-epimer 25(OH)D
molecules.9
2.2. Principles of the ADVIA Centaur
Vitamin D Total assay procedure
The ADVIA Centaur Vitamin D Total
assay is a one-pass, 18-minute competitive
immunoassay that uses an anti-fluorescein
monoclonal mouse antibody covalently
bound to paramagnetic particles, an anti-
25(OH)D monoclonal mouse antibody
labeled with acridinium ester (AE), and a
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │3
vitamin D analog labeled with fluorescein.
The assay demonstrates equimolar cross-
reactivity with 25(OH)D3 (100.7%) and
25(OH)D2 (104.5%), minimal cross-reactivity
with 3-epimer-25(OH)D3 (1.1%), and a broad
dynamic range of 4.2 to 150 ng/mL (10.5 to
375 nmol/L). Other performance characteris-
tics were not altered by the standardization.
Evaluation shows that the limit of blank
= 1.7 ng/mL, limit of detection = 3.2 ng/mL,
and limit of quantitation = 4.2 ng/mL. The
assay also demonstrated good linearity and
dilution recovery in internal studies (data not
shown).
2.3. Alignment with the VDSP NIST-
Ghent RMP
Using the ADVIA Centaur Vitamin D
Total assay master curve, dose values were
determined for 177 native human serum
samples and compared with dose values
assigned by the VDSP NIST-Ghent RMP
methods. The latter values spanned most of
the assay range (5–140 ng/mL), 90 samples
were assigned by either NIH or CDC, 60
samples were assigned by a commercial
vendor using an LC-MS/MS method aligned
to the VDSP NIST-Ghent RMP, and 27
samples were value-assigned by Ghent
University. Two reagent lots (R1 and R2)
were used in conjunction with their respective
calibrator lots (C1 and C2) on a single
ADVIA Centaur XP system. Deming
regression was used to compare results from
the ADVIA Centaur Vitamin D Total assay to
dose values determined using the VDSP
NIST-Ghent RMP.9,10
A second evaluation was conducted
using 122 additional serum samples
comprising 116 native samples (range
7.8 ng/mL to 148.1 ng/mL) and six samples
that were created by spiking a serum sample
with 25(OH)D2 to achieve a concentration
range of 70.4 ng/mL to 125.6 ng/mL. All
samples were assayed in singlicate on a single
ADVIA Centaur system using a single
ADVIA Centaur Vitamin D Total assay
reagent lot (R3), and values were compared
to dose values determined using the VDSP
RMP at Ghent University using Deming
regression and Bland-Altman analyses.
2.4. Comparison to College of
American Pathologists (CAP) and Vitamin
D External Quality Assessment Scheme
(DEQAS) survey samples
CAP 2011 accuracy-based vitamin D
(ABVD) and DEQAS survey samples
(n = 30) were measured using two reagent
lots, lot 1 (R1) and lot 2 (R2) and the ADVIA
Centaur Vitamin D Total assay. Percent bias
to value assignments made using the VDSP
NIST-Ghent RMP (range = 14.1 ng/mL to
58.5 ng/mL) was determined.
Percent bias = [(ADVIA Centaur Vitamin D
Total Assay Dose – Survey-assigned
Dose)/Survey-assigned Dose] x 100
In addition, DEQAS survey samples
available from 2012 through January 2015
were evaluated using a single lot of the
ADVIA Centaur Vitamin D Total assay and
results (n = 50) were compared to the
DEQAS-all-laboratory trimmed mean
(ALTM) in 2012. Beginning in April 2013,
DEQAS began using the VDSP NIST-Ghent
RMP to assign values to its survey samples
and as the target value for determining
percent bias (rather than the all-laboratory
trimmed mean [ALTM]).
The CAP and DEQAS criterion for total
vitamin D is that 80% of results should fall
within 25% of the target value.
2.5. Imprecision
Imprecision (%CV) was performed
according to the CLSI EP15-A2 guideline for
verification of performance and precision.14
Two human serum samples (<20ng/mL) and
four pooled samples prepared from
25(OH)D3-spiked human serum
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │4
(~20 to ~130 ng/mL) were run over 20 days,
two runs per day, using two lots, lot 1 (R1)
and lot 2 (R2), and two replicates per run on
two ADVIA Centaur XP platforms
(n = 80/sample). The VDSP NIST-Ghent
RMP reagent master curves and calibrator
assignments were used to evaluate the 20-day
imprecision data to calculate reproducibility
and within-lab CV.
2.6. Performance evaluation using
total analytical error (TAE)
In a separate study, TAE and %TAE
were calculated from overall precision error
(standard deviation [SD] and bias), and used
to determine acceptability of performance.
Two lots of reagents (R2 and R3) were used
to measure one high and one low calibrator,
and four pooled samples that were different
from those used in the precision study
(PS A - PS D) in quadruplicate across three
ADVIA Centaur systems, once daily for 4
days (n/sample = 144). PS A contained no
25(OH)D3 spiking agent (approximate final
25[OH]D3 concentration = 20 ng/mL).
Spiking agent was used to adjust the final
25(OH)D3 concentration in the other pooled
samples: PS B = 50 ng/mL,
PS C = 120 ng/mL, PS D = 50 ng/mL. In
addition, two internal standard level control
values assigned using the ADVIA Centaur
Vitamin D Total assay, and two internal
control lots were evaluated using each
reagent lot. Value assignments were made for
each sample following full curve analysis.
Mean concentration was determined for each
sample following calibration, and the SD was
used to calculate the precision error. Bias
between each value assignment and the mean
concentration of each sample was determined
and used to calculate TAE and %TAE:
TAE = |Total bias| + 2SD
%TAE = (TAE/assigned value)x100%.15
2.7. CDC Vitamin D Standardization
Certification Program (VDSCP) studies
The CDC VDSCP is a two-phase
process. To become VDSCP certified,
participants must pass four consecutive
quarterly challenges using blinded samples
after an initial calibration period (Phase I).
Samples for both phases of the study are
value-assigned using the VDSP NIST-Ghent
RMP and supplied by the CDC. Results are
analyzed by the CDC according to CLSI
document EP9-A2 and used to determine
bias, precision, and total error.16,17
Following
the calibration period, 10 individual patient
samples supplied by the CDC and containing
blinded quantities of 25(OH)D-total were
analyzed in duplicate over 2 days (n = 40)
(Phase 2). Four quarterly blinded assessments
were conducted over the course of 2013 (once
quarterly) for a total of 160 replicates. Four
quarterly assessments were also performed in
subsequent years. The criterion for achieving
CDC VDSCP certification is a mean bias for
all 40 samples (160 results) of ±5% to the
CDC and University of Ghent Vitamin D2
and D3 Reference Method, and an overall
imprecision of <10%.18
Although the CDC
normally supplies 40 samples for the initial
calibration phase, 50 were sent to Siemens
and tested.16
2.8. Alignment to a third-party
VDSCP-certified method
The ADVIA Centaur Vitamin D Total
assay was compared to the Endocrine
Sciences Laboratory CDC VDSCP-certified
ID-LC/MS/MS method (Esoterix, Endocrine
Sciences, Calabasas Hills, CA; a LabCorp
Specialty Testing Group Member).19
Samples
(n = 149) were tested in singlicate using a
single ADVIA Centaur reagent lot (R4). After
testing, samples across the range of the assay
were selected and sent to Esoterix for testing
using their CDC VDSCP-certified ID-
LC/MS/MS method.
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │5
3. Results
3.1. Alignment with the VDSP NIST-
Ghent RMP
Deming regression analysis was
performed for 177 samples in study 1 to
compare the ADVIA Centaur Vitamin D
Total assay results to the VDSP NIST-Ghent
RMP-generated values. A correlation of 96%
and slope of 0.99 for both reagent lots was
demonstrated between the ADVIA Centaur
Vitamin D Total assay results and the ID-
LC/MS/MS method. A small negative bias
was observed with R2 but was not observed
with R1 (Figures 1A and 1B). In the second
study with 122 samples, Deming regression
demonstrated similar results as Study 1
(Figure 1C). Bland-Altman analysis
demonstrated a mean bias of 0.09 ng/mL
(Figures 1C and 1D). Although greater bias
was observed at higher concentrations, with
the exception of a few outliers, bias did not
exceed 12 ng/mL.
Figure 1.
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │7
Figure 1. Deming regression analysis comparing the ADVIA Centaur Vitamin D Total assay for
177 samples with values assigned using methods aligned with the NIST–Ghent RMP for
(A) R1 and (B) R2; (C) Deming regression and (D) Bland Altman analysis comparing the
ADVIA Centaur Vitamin D Total assay for 122 samples with values assigned directly at Ghent
University using their RMP, R3. To convert 25(OH)D concentrations to nanomoles per liter
(nmol/L), multiply by 2.5.
3.2. Comparison to CAP and DEQAS
survey samples
The CAP and DEQAS acceptance
requirement for total vitamin D is that 80% of
results fall within 25% of the assigned target
value. For the 2011 samples, all but one of
the ADVIA Centaur Vitamin D Total assay
results (n = 30) were within 25% of the
survey results, although some inconsistencies
in percent bias between samples and between
lots were observed (Table 1). Greater than
half of the results were within 10% (eight of
the 15 samples in R1 and ten of the 15
samples in R2).
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Table 1. Comparison of R1 and R2 results with CAP and DEQAS survey results.
Sample
Survey-
Assigned
Value
ADVIA
Centaur
RMP, Lot
R1
(ng/mL)*
ADVIA
Centaur
RMP, Lot
R2
(ng/mL)
Bias to
Assigned
Value, Lot
R1
Bias to
Assigned
Value, Lot
R2
DEQAS
(ID-
LC/MS/MS)
421 24.0 23.1 24.1 -3.7% 0.4%
422 15.8 16.3 16.6 2.9% 5.2%
423 35.9 31.6 31.8 -12.0% –11.3%
424 19.6 21.5 23.0 9.8% 17.1%
425 19.6 19.1 19.8 -2.6% 1,0%
426 14.1 13.8 16.8 -2.0% 19.2%
427 32.1 26.2 29.2 -18.3% –9.1%
428 22.1 19.2 20.1 -13.0% –9.1%
429 24.2 23.8 24.2 -1.5% 0.1%
430 17.6 21.2 23.1 20.3% 31.4%
CAP
ABVD
1 20.80 22.29 25.14 7.2% 20.4%
ABVD
2 14.60 14.14 14.56 -3.2% –0.3%
ABVD
3 32.60 27.88 29.51 -14.5% –9.5%
ABVD
4 58.50 50.06 53.71 -14.4% –8.2%
ABVD
5 39.90 39.20 41.13 -1.7% 3.1%
*To convert 25(OH)D concentrations to nanomoles per liter (nmol/L), multiply by 2.5. CAP:
College of American Pathologists; DEQAS: Vitamin D External Quality Assessment Scheme;
ID-LC/MS/MS: Isotope dilution liquid chromatography mass spectrometry; ABVD:
accuracy-based vitamin D survey samples.
When compared to the VDSP NIST-
Ghent RMP for DEQAS survey sample 421–
470, the smallest percent bias observed was
0.2%, while the greatest percent bias was
45.2%. Percent bias > 25% (the maximum
allowable by DEQAS) was observed for only
four of the 50 DEQAS samples (8%). Median
percent bias was 1.7% for all results, and
most samples were within 10% of the
assigned value (Figure 2).
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Figure 2. Distribution of percent bias for all 50 DEQAS samples (2012–2015).
3.3. Imprecision
The VDSCP requires <10% impreci-
sion (CV).20
To assure that was achieved,
tolerance for repeatability variance was <8%
for samples between 20 and 30 ng/mL
(50–75 nmol/L) and <7% for samples
between >30 to 150 ng/mL (>75 to
374 nmol/L). Within run specifications were
met, although the total %CV for one sample
using one lot on two systems was slightly
greater than the maximum acceptable
(Table 2).
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ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Table 2. Precision study results for the standardized ADVIA Centaur Vitamin D Total assay
for individual samples (SHS1 and SHS2) and pooled sera (PS1–4), using R1 and R2.
Within Run Total
System Lot Sample Mean
(ng/mL)* SD CV SD CV
A
R1
SHS1 13.60 0.64 4.7 1.61 11.9
SHS2 17.22 0.91 5.3 1.70 9.9
PS 1 28.20 1.45 5.2 2.02 7.2
PS 2 46.08 1.79 3.9 2.79 6.1
PS 3 73.22 2.71 3.7 4.36 6.0
PS 4 114.08 3.44 3.0 4.77 4.2
R2
SHS1 11.90 0.45 3.8 0.95 8.0
SHS2 15.26 0.56 3.7 1.35 8.9
PS 1 23.25 1.19 5.1 1.88 8.1
PS 2 41.45 1.48 3.6 2.22 5.3
PS 3 69.00 3.11 4.4 4.10 5.9
PS 4 112.25 3.47 3.1 4.22 3.8
B
R1
SHS1 15.17 0.61 4.0 1.98 13.0
SHS2 19.77 0.90 4.6 1.76 8.9
PS 1 31.00 1.30 4.2 2.36 7.6
PS 2 50.26 1.83 3.6 3.94 7.8
PS 3 78.95 3.37 4.3 6.99 8.9
PS 4 120.31 1.99 1.7 5.25 4.4
R2
SHS1 14.30 0.71 5.0 1.31 9.1
SHS2 17.90 0.98 5.5 1.47 8.2
PS 1 25.34 1.49 5.9 1.69 6.7
PS 2 45.79 2.40 5.2 3.72 8.1
PS 3 74.83 3.53 4.7 6.39 8.5
PS 4 117.34 2.94 2.5 4.81 4.1
*To convert 25(OH)D concentrations to nanomoles per liter (nmol/L), multiply by 2.5. SD:
Standard deviation. CV: Coefficient of variation.
3.4. Performance evaluation using
total analytical error (TAE)
According to calculations by Stöckl et
al., total error based on the DEQAS limit for
error should be less than 46%.20
Siemens
chose more stringent criteria, requiring
%TAE to be less than ±45% for samples
between 2 and 20 ng/mL, and less than ±35%
for samples over 20 ng/mL. These
specifications were met for all samples
(Table 3).
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Table 3. Total analytical error (TAE) for samples with assigned values between 17 and
121 ng/mL, using R2 and R3.
Sample
VAa
(ng/mL)
*
Average
Dose
(ng/mL)* Bias
Observed
SD Total
Observed
CV Total
TAE
(|Bias|
+ 2SD) %TAE
Lot 1 high
calibrator 116.54 118.36 1.81 4.85 4.1 11.5 9.9
Lot 1 low
calibrator 28.43 28.31 -0.12 3.77 13.3 7.7 26.9
Lot 2 high
calibrator 104.01 103.05 -0.97 5.02 4.9 11.0 10.6
Lot 2 low
calibrator 23.33 23.62 0.29 3.64 15.4 7.6 32.5
Ctrl lot 1 25.18 25.32 0.15 3.51 13.9 7.2 28.5
Ctrl lot 2
100.30 98.81 -1.49 6.61 6.7 14.7 14.7
PS A 25.92 26.01 0.09 3.26 12.5 6.6 25.5
PS B 53.56 51.61 -1.95 3.61 7.0 9.2 17.1
PS C 120.88 124.36 3.48 4.27 3.4 12.0 9.9
PS D 52.39 50.51 -1.88 3.51 6.9 8.9 17.0
Internal
standardb
17.93 17.84 -0.09 2.35 13.2 4.8 26.7
aValue assignment.
bInternal standards have values assigned by method comparison to the
University of Ghent RMP. *To convert 25(OH)D concentrations to nanomoles per liter (nmol/L),
multiply by 2.5. PS: Pooled sera; SD: Standard deviation; CV: Coefficient of variation.
3.5. CDC Vitamin D Standardization
Certification Program (VDSCP) studies
The correlation between the CDC
VDSP NIST-Ghent RMP-assigned values and
the ADVIA Centaur Vitamin D Total assay
values was strong across all four quarterly
blind trials conducted in 2013 (Figure 3). The
mean bias was 0.3% (SD = 16.8,
95% CI = –5.0 to 5.6), the mean CV was
5.5% (SD = 3.2, 10th percentile = 1.6%,
90th percentile = 9.8%), and 86% of samples
in the challenge groups were within the
suggested total error of ±21.5%. Similar
results were obtained across all four quarterly
trials conducted for each consecutive
subsequent year to date (data not shown).
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Figure 3. (A) Linear regression and (B) total error generated by CDC VDSCP from all challenge
samples in 2013. To convert 25(OH)D concentrations to nanograms per mL (ng/mL), divide by
2.5.
x
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3.6. Alignment to a third-party
VDSCP-certified method
The ADVIA Centaur Vitamin D Total
assay aligned well with the Esoterix method
(n = 149) as demonstrated by both Deming
regression (Pearson’s r = 0.95) and
Bland–Altman analysis (bias = 1.13) (Figure
4A and B). The Esoterix assay, which is
capable of differentiating between the 2
primary forms of 25(OH)D, identified 55
samples containing 25(OH)D2.19
The two
assays were harmonized regardless of
whether the sample contained both 25(OH)D2
and 25(OH)D3 (n = 55, Pearson’s r = 0.95,
bias = 2.6) (Figure 4C and 4D) or 25(OH)D3
only (n = 94, Pearson’s r = 0.97, bias = 0.26)
(Figures 4E and 4F).
Figure 4.
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
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Figure 4. Alignment was demonstrated between the Siemens ADVIA Centaur Vitamin D Total
immunoassay and the Esoterix ID-LC/MS/MS Vitamin D method for 149 samples. (A) Deming
regression and (B) Bland-Altman analysis for all 149 samples for total vitamin D; C–F: Deming
regression and Bland-Altman analysis, comparing the Esoterix and Siemens assays for those 55
samples containing both 25(OH)D2 and 25(OH)D3 (C and D) and the 94 samples containing
25(OH)D3 alone (E and F). To convert 25(OH)D concentrations to nanomoles per liter (nmol/L),
multiply by 2.5.
4. Discussion
As pointed out by the U.S. Office of
Dietary Standards, variability between
25(OH)D assays has hampered efforts to
compare vitamin D levels across studies as
well as the development of diagnostic cut
points and therapeutic guidelines.11,13
Standardization of assays would alleviate
these problems.9,11,13
In this study, we report
standardization and continued adherence of
the Siemens ADVIA Centaur Vitamin D
Total assay to the VDSP NIST-Ghent RMP.
The previous calibration for the
ADVIA Centaur Vitamin D Total assay was
observed to be positively biased when
compared to both the VDSP NIST-Ghent
RMP and the ADVIA Centaur Vitamin D
Total recalibration (data not shown). Previous
studies have found that the ADVIA Centaur
Vitamin D Total assay over-reported vitamin
D levels.21
Those previous studies were
performed using the non-VDSP NIST-Ghent
RMP standardized version of the Siemens
assay and would need to be repeated using
the standardized assay. Additionally,
concentration-dependent variability of
vitamin D binding protein has been reported
when using the original Siemens assay.5 The
authors commented that alignment to the
VDSP NIST-Ghent RMP (which at the time
was still under consideration) should resolve
the issue. A subsequent study using the
standardized ADVIA Centaur Vitamin D
Total assay was found to accurately measure
vitamin D in samples from different
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │17
populations with elevated vitamin D binding
protein levels.22
In the present report, precision and
TAE performance acceptability criteria were
fulfilled for the standardized ADVIA Centaur
Vitamin D Total assay. Regression and bias
analyses of two sample sets confirm that the
standardized ADVIA Centaur Vitamin D
Total assay correlates well with the VDSP
NIST-Ghent RMP for directly assigned
values and also for values from
metrologically traceable methods. DEQAS
and CAP survey results support this claim:
for R1 of the standardized assay, 100% of the
results were within 25% of both the CAP and
DEQAS samples, and 97% of results for R2
were within 25% of the CAP and DEQAS
established values, demonstrating that the
assay met the CAP and DEQAS proficiency
testing criteria. Furthermore, 86% of samples
tested with R1 and 80% of the samples tested
with R2 yielded values that were within
15.8% of the survey-assigned values, which
has been reported to be the allowable limit for
assay bias.6 The Esoterix assay was
independently standardized to the VDSP
NIST-Ghent RMP and independently
certified by the VDSCP. The alignment to the
Esoterix assay provided further evidence that
the ADVIA Centaur Vitamin D Total assay
was standardized to the VDSP NIST-Ghent
RMP. Correlation of the ADVIA Centaur
Vitamin D Total assay with the Esoterix ID-
LC/MS/MS assay of 25(OH)D2-containing
samples, along with detection of 25(OH)D2-
spiked samples used in the TAE performance
evaluation study, show that the assay detected
both 25(OH)D2 and 25(OH)D3 with equal
reliability.
The CDC VDSCP program was
initiated as an adjunct program to the VDSP
to establish a protocol for certifying that
vitamin D assays remain standardized to the
VDSP NIST-Ghent RMP. Participants must
pass four consecutive quarterly yearly
challenges using blinded samples: Mean bias
for all 40 samples (160 results) must be ±5%
of the CDC values and overall imprecision
must be <10%.20
Certification must be
renewed annually. The standardized ADVIA
Centaur Vitamin D Total assay performed
appreciably better than the criteria, becoming
one of the first assays to be certified by the
CDC, one of only three immunoassays
certified as of February 2014 as part of the
initial certification program, and to our
knowledge, the only fully automated assay to
become certified for four consecutive years
(2014– 2017).
5. Conclusion
The CDC VDSCP certification attests
to the performance of the Siemens ADVIA
Centaur Vitamin D Total assay and its
adherence to the VDSP. This certification, in
conjunction with the performance and method
comparison data indicate that the Siemens
ADVIA Centaur Vitamin D Total assay,
traceable to the VDSP NIST-Ghent RMP,
meets the acceptance criteria for use in the
clinical laboratory.
Disclosure
All authors are employees of Siemens
Healthineers.
Conflicts of interest:
The authors declare that there are no
conflicts of interest regarding the publication
of this paper. All authors are employees of
Siemens Healthineers.
Acknowledgments:
This study was supported by Siemens
Healthineers. The authors would like to thank
V. Shalhoub, PhD for help with this
manuscript.
Medical Research Archives, Vol. 5, Issue 8, August 2017
ADVIA Centaur Vitamin D Total Assay Standardization/Certification
Copyright 2017 KEI Journals. All Rights Reserved Page │18
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